Characteristics of the gut microbiota of patients with symptomatic carotid atherosclerotic plaques positive for bacterial genetic material.

Background: The gut microbiota (GM) is believed to be closely associated with symptomatic carotid atherosclerosis (SCAS), yet more evidence is needed to substantiate the significant role of GM in SCAS. This study, based on the detection of bacterial DNA in carotid plaques, explores the characteristics of GM in SCAS patients with plaque bacterial genetic material positivity, aiming to provide a reference for subsequent research. Methods: We enrolled 27 healthy individuals (NHF group) and 23 SCAS patients (PFBS group). We utilized 16S rDNA V3-V4 region gene sequencing to analyze the microbiota in fecal samples from both groups, as well as in plaque samples from the carotid bifurcation extending to the origin of the internal carotid artery in all patients. Results: Our results indicate significant differences in the gut microbiota (GM) between SCAS patients and healthy individuals. The detection rate of bacterial DNA in plaque samples was approximately 26%. Compared to patients with negative plaques (PRSOPWNP group), those with positive plaques (PRSOPWPP group) exhibited significant alterations in their GM, particularly an upregulation of 11 bacterial genera (such as Klebsiella and Streptococcus) in the gut, which were also present in the plaques. In terms of microbial gene function prediction, pathways such as Fluorobenzoate degradation were significantly upregulated in the GM of patients with positive plaques. Conclusion: In summary, our study is the first to identify significant alterations in the gut microbiota of patients with positive plaques, providing crucial microbial evidence for further exploration of the pathogenesis of SCAS.

Laboratory Grown Biofilms of Bacteria Associated with Human Atherosclerotic Carotid Arteries Release Collagenases and Gelatinases during Iron-Induced Dispersion.

The association of bacteria with arterial plaque lesions in patients with atherosclerosis has been widely reported. However, the role these bacteria play in the progression of atherosclerosis is still unclear. Previous work in our lab has demonstrated that bacteria exist in carotid artery plaques as biofilm deposits. Biofilms are communities of microorganisms enmeshed within a protective, self-produced extracellular matrix and have been shown to contribute to chronic infections in humans. Biofilm communities have the potential to impact surrounding tissues in an infection if they undergo a dispersion response, releasing bacteria into the surrounding environment by enzymatic degradation of the extracellular matrix. One concern relating to these enzymes is that they could cause collateral damage to host tissues. In this study, we present an in vitro multispecies biofilm culturing model used to investigate the potential role of bacterial biofilm dispersion in the progression of atherosclerosis. This work has demonstrated an increase in cell release from mixed-species biofilms formed by bacteria associated with human carotid arterial plaque deposits following treatment with iron or a combination of norepinephrine and transferrin. Greater extracellular lipase, protease, and collagenase/gelatinase activity was also associated with iron-treated biofilms. The results of this work suggest that bacteria in this model undergo iron-induced biofilm dispersion, as evidenced by the increased cell release and higher enzyme activity following treatment. This work demonstrates the potential for multispecies biofilm dispersion to contribute to arterial tissue degradation by bacteria and suggests that in atherosclerotic infections, biofilm dispersion may contribute to thrombogenesis, which can lead to heart attack or stroke. IMPORTANCE Atherosclerosis, or hardening of the arteries, is a leading cause of congestive heart failure, heart attack, and stroke in humans. Mounting evidence, in the literature and from our lab, points to the regular involvement of bacteria within arterial plaque deposits in patients with advanced atherosclerosis. Very little is known about the behavior of these bacteria and whether they may contribute to tissue damage in infected arteries. Tissue damage within the arterial plaque lesion can lead to rupture of the plaque contents into the bloodstream, where a clot may form, resulting in a potential heart attack or stroke. This study shows that plaque-associated bacteria, when cultured as mixed-species biofilms in the laboratory, can release degradative enzymes into their environment as the result of a dispersion response triggered by iron. These degradative enzymes can digest proteins and lipids which are associated with the tissues that separate the plaque lesion from the arterial lumen. Thus, this study demonstrates that if mixed species biofilms are induced to undergo dispersion in an infected atherosclerotic lesion when exposed to an elevated concentration of free iron, they have the potential to contribute to the weakening of arterial tissues, which may contribute to atherosclerotic plaque destabilization.

Identification of Periopathogens in Atheromatous Plaques Obtained from Carotid and Coronary Arteries.

Increasing attention has been paid to the possible link between periodontal disease and atherosclerosis over the past decade. The aim of this study is to investigate the presence of five periopathogens: Porphyromonas gingivalis (P.g.), Aggregatibacter actinomycetemcomitans (A.a.), Tannerella forsythia (T.f.), Treponema denticola (T.d.), and Prevotella intermedia (P.i.) in atheromatous plaques obtained from the carotid and coronary arteries in patients who underwent coronary artery bypass graft surgery and carotid endarterectomy. Group I (carotid arteries) consisted of 30 patients (mean age: 54.5 ± 14.8), and group II (coronary arteries) consisted of 28 patients (mean age: 63 ± 12.1). Clinical periodontal examinations consisted of plaque index, gingival index, sulcus bleeding index, and periodontal probing depth and were performed on the day of vascular surgery. The presence of periopathogens in periodontal pockets and atherosclerotic vessels was detected using polymerase chain reaction. In both subgingival plaque and atherosclerotic plaque of carotid arteries, P.g., A.a., T.f., T.d., and P.i. were detected in 26.7%, 6.7%, 66.7%, 10.0%, and 20.0%, respectively, while for coronary arteries, P.g. was detected in 39.3%, A.a. in 25%, T.f. in 46.4%, T.d. in 7.1%, and P.i. in 35.7%. The presence of five periopathogens in carotid and coronary atherosclerotic vessels showed correlation in regard to the degree of periodontal inflammation. The present study suggests the relationship between periodontal pathogenic bacteria and atherogenesis. Further studies are necessary in relation to the prevention or treatment of periodontal disease that would result in reduced mortality and morbidity associated with atherosclerosis.

Guttural pouch mycosis, sympathy for Aspergillus nidulans / Micosis de las bolsas guturales: simpatía por Aspergillus nidulans

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Common carotid arteritis and polymyalgia with Mycoplasma pneumoniae infection.

A few pediatric cases with brain vasculitis most frequently affecting the middle cerebral artery have been reported in association with Mycoplasma pneumoniae infection, but involvement of the common carotid artery (CCA) before the bifurcation has not been reported to date. We report herein a case of 10-year-old boy with common carotid arteritis and polymyalgia associated with Mycoplasma pneumoniae infection. His fever and cough began 2 weeks before, and his right upper and lower extremity pains began 2 days before admission. He had initially been treated with clarithromycin followed by tosufloxacin, but his symptoms persisted. His M. pneumonia-specific antibody titer was high on admission (1:10240 by particle agglutination method) and the gene of M. pneumoniae was detected in a throat swab specimen by the loop-mediated isothermal amplification method with initial high levels of serum interleukin-8, tumor necrosis factor-α, and interleukin-18 along with elevated blood levels of complements. On the 5th day of hospitalization, vascular echograms of the extremities and neck showed increasing intima-media thickness of bilateral CCAs without stenosis and/or thrombosis and T2-weighted with lipid suppression magnetic resonance imaging of the neck showed high signal intensity of bilateral CCA walls. Coagulation studies were unremarkable and no autoantibodies were detected as far as tested. He was successfully treated by intravenous administration of prednisolone and was stable without any neurological sequelae 17 months after the onset without medication. His particle agglutination titer decreased to 1:5120, and serum interleukin-8, tumor necrosis factor-α, interleukin-18, and complement levels returned to normal.

[Establishment of rat model with diabetes mellitus and concomitant periodontitis and the carotid artery lesions in the model rats].

Objectives: To establish SD rat model with type 2 diabetes mellitus (DM) and concomitant chronic periodontitis (CP) and to evaluate the influence of periodontitis on the vascular lesions of type 2 diabetes rats. Methods: Totally 241 clean level SD rats were randomly divided into four groups, group A (normal control, NC, n=27), group B (DM, n=34), group C (CP, n=90) and group D (DM+CP, n=90). The rats of DM group were fed with high-fat and high-sugar diet for 8 to 10 weeks, and then were multiply injected with small dose streptozotocin under the condition of ice bath. Blood sugar levels after the injection were dynamically monitored at 72 h, 1 week, 2 weeks and 4 weeks, respectively. The CP model was established by means of ligation. Bilateral maxillary first and second molars were selected and ligated using 0.2 mm orthodontic wires binding with 4-0 surgical suture soaked with Porphyromonas gingivalis (Pg) suspension. After a period of 14 weeks, all the rats were put to death. Maxillary samples were subjected to methylene blue staining to observe alveolar bone loss. Bilateral carotid artery specimens were collected. The left carotid artery specimens were used to detect the prevalence of Pg using quantitative real-time PCR. The right carotid artery specimens were used to observe pathological changes. Results: Blood sugar levels of rats in group B and D increased and changed sharply after Streptozotocin injection with in 1 week. Symptoms of 'more drink, more food and body weight loss' appeared. The fasting blood glucose (FBG) was more than 7.8 mmol/L and (or) the random blood glucose (RBG) was more than 17.8 mmol/L. Both FBG and RBG became stable after 2 to 3 weeks. Levels of HbA1C in group B and D ([7.32±0.45]%, [9.41±0.45]%) were significantly higher than that of group A ([4.02±0.45]%) (P<0.01). Rats of group D were observed the most severe bone loss showing wider interdental space and furcation involvement. Pathological results of carotid artery tissues of group D showed the worst lesions including thinning and calcification of vessel walls, and breaking down or disappearance of elastic fibers. The prevalences of DNA of Pg in groups of A, B, C and D were 3/7, 3/7, 6/7 and 7/7, respectively. The bacteria numbers detected by quantitative real-time PCR in groups C and D were significantly higher than that of groups A and B (P<0.01). Conclusions: Rat model of type 2 DM with periodontitis was successfully established in the present study. Carotid artery specimens from DM+CP model rats showed typical vascular lesions such as calcification and fiber disorders. Pg was found in all carotid specimens and the highest bacteria numbers were detected in the composite model rats. The Pg might play a role in the progress of diabetes vascular lesions.

Deposition and hydrolysis of serine dipeptide lipids of Bacteroidetes bacteria in human arteries: relationship to atherosclerosis.

Multiple reaction monitoring-MS analysis of lipid extracts from human carotid endarterectomy and carotid artery samples from young individuals consistently demonstrated the presence of bacterial serine dipeptide lipid classes, including Lipid 654, an agonist for human and mouse Toll-like receptor (TLR)2, and Lipid 430, the deacylated product of Lipid 654. The relative levels of Lipid 654 and Lipid 430 were also determined in common oral and intestinal bacteria from the phylum Bacteroidetes and human serum and brain samples from healthy adults. The median Lipid 430/Lipid 654 ratio observed in carotid endarterectomy samples was significantly higher than the median ratio in lipid extracts of common oral and intestinal Bacteroidetes bacteria, and serum and brain samples from healthy subjects. More importantly, the median Lipid 430/Lipid 654 ratio was significantly elevated in carotid endarterectomies when compared with control artery samples. Our results indicate that deacylation of Lipid 654 to Lipid 430 likely occurs in diseased artery walls due to phospholipase A2 enzyme activity. These results suggest that commensal Bacteriodetes bacteria of the gut and the oral cavity may contribute to the pathogenesis of TLR2-dependent atherosclerosis through serine dipeptide lipid deposition and metabolism in artery walls.

Bacterial profile in human atherosclerotic plaques.

BACKGROUND AND AIMS: Several studies have confirmed the presence of bacterial DNA in atherosclerotic plaques, but its contribution to plaque stability and vulnerability is unclear. In this study, we investigated whether the bacterial plaque-profile differed between patients that were asymptomatic or symptomatic and whether there were local differences in the microbial composition within the plaque. METHODS: Plaques were removed by endarterectomy from asymptomatic and symptomatic patients and divided into three different regions known to show different histological vulnerability: A, upstream of the maximum stenosis; B, site for maximum stenosis; C, downstream of the maximum stenosis. Bacterial DNA composition in the plaques was determined by performing 454 pyrosequencing of the 16S rRNA genes, and total bacterial load was determined by qPCR. RESULTS: We confirmed the presence of bacterial DNA in the atherosclerotic plaque by qPCR analysis of the 16S rRNA gene but observed no difference (n.s.) in the amount between either asymptomatic and symptomatic patients or different plaque regions A, B and C. Unweighted UniFrac distance metric analysis revealed no distinct clustering of samples by patient group or plaque region. Operational taxonomic units (OTUs) from 5 different phyla were identified, with the majority of the OTUs belonging to Proteobacteria (48.3%) and Actinobacteria (40.2%). There was no difference between asymptomatic and symptomatic patients, or plaque regions, when analyzing the origin of DNA at phylum, family or OTU level (n.s.). CONCLUSIONS: There were no major differences in bacterial DNA amount or microbial composition between plaques from asymptomatic and symptomatic patients or between different plaque regions, suggesting that other factors are more important in determining plaque vulnerability.

Bacteria of leg atheromatous arteries responsible for inflammation.

BACKGROUND: Ischaemia of the lower limbs is frequently followed by inflammation and, in advanced cases, necrosis of peripheral tissues. Whether this is caused by arterial hypoperfusion only or by the presence of bacteria in the arterial walI as well remains unclear. The aim of the study was to prove the presence and source of bacteria in arterial specimens and evaluate their chemotactic properties resulting in the formation of periarterial cellular infiltrates. MATERIALS AND METHODS: Bacterial culture and testing for 16sRNA were performed in fragments of popliteal artery harvested from amputated limbs. Carotid artery plaques served as controls. Fragments of arteries were transplanted into scid mice to evaluate their chemotactic activity for macrophages. RESULTS: a) higher prevalence of isolates and 16sRNA in atherosclerotic popliteal than carotid arteries, b) high density of plaque and periarterial infiltrates and mRNA level for pro-inflammatory cytokines in popliteal arteries, c) prevalent microbes were Staphylococcus aureus, S. epidermidis and Enterococci, d) foot skin and arterial bacterial phenotypes and DNA revealed evident similarities, and e) more intensive mouse macrophage accumulation in popliteal than carotid implants into scid mice. CONCLUSIONS: The presence of bacteria in the lower limb arterial wall was documented. They may predispose to inflammation secondary to ischaemic changes.

Presence of Periodontopathic Bacteria DNA in Atheromatous Plaques from Coronary and Carotid Arteries.

OBJECTIVES: Interest in periodontitis as a potential risk factor for atherosclerosis and its complications resulted from the fact that the global prevalence of periodontal diseases is significant and periodontitis may induce a chronic inflammatory response. Many studies have analyzed the potential impact of the Porphyromonas gingivalis, major pathogen of periodontitis, on general health. The purpose of this study was to find the presence of the Porphyromonas gingivalis DNA in the atherosclerotic plaques of coronary and carotid arteries and in the periodontal pockets in patients with chronic periodontitis, who underwent surgery because of vascular diseases. METHODS AND RESULTS: The study population consisted of 91 patients with coronary artery disease or scheduled for carotid endarterectomy. The presence of Porphyromonas gingivalis DNA in atheromatous plaques and in subgingival samples was determined by PCR. Bacterial DNA was found in 21 of 91 (23%) samples taken from vessels and in 47 of 63 (74.6%) samples from periodontal pockets. CONCLUSIONS: Porphyromonas gingivalis DNA is frequently found in atheromatous plaques of patients with periodontitis. That is why more research should be conducted to prove if this periopathogen may have an impact on endothelium of patients at risk of atherosclerosis.

Propionibacterium acnes Recovered from Atherosclerotic Human Carotid Arteries Undergoes Biofilm Dispersion and Releases Lipolytic and Proteolytic Enzymes in Response to Norepinephrine Challenge In Vitro.

In the present study, human atherosclerotic carotid arteries were examined following endarterectomy for the presence of the Gram-positive bacterium Propionibacterium acnes and its potential association with biofilm structures within the arterial wall. The P. acnes 16S rRNA gene was detectable in 4 of 15 carotid artery samples, and viable P. acnes was one among 10 different bacterial species recoverable in culture. Fluorescence in situ hybridization analysis of 5 additional atherosclerotic carotid arteries demonstrated biofilm bacteria within all samples, with P. acnes detectable in 4 samples. We also demonstrated that laboratory-grown cultures of P. acnes biofilms were susceptible to induction of a biofilm dispersion response when challenged with physiologically relevant levels of norepinephrine in the presence of iron-bound transferrin or with free iron. The production and release of lipolytic and proteolytic extracellular enzymes by P. acnes were shown to increase in iron-induced dispersed biofilms, and these dispersion-induced P. acnes VP1 biofilms showed increased expression of mRNAs for the triacylglycerol lipases PPA2105 and PPA1796 and the hyaluronate lyase PPA380 compared to that in untreated biofilms. These results demonstrate that P. acnes can infect the carotid arteries of humans with atherosclerosis as a component of multispecies biofilms and that dispersion is inducible for this organism, at least in vitro, with physiologically relevant levels of norepinephrine resulting in the production and release of degradative enzymes.

The presence of some cytokines and Chlamydia pneumoniae in the atherosclerotic carotid plaque in patients with carotid artery stenosis.

BACKGROUND: Over the last few years the role of microorganisms in the pathogenesis of atherosclerosis has been widely discussed. Chlamydia pneumoniae activates immune cells to produce cytokines that are responsible for the formation of atheromatous carotid lesions. MATERIAL AND METHODS: The study was carried out at the Department of Vascular, General and Transplantation Surgery, Wroclaw Medical University, in 2002-2003, on 100 consecutive symptomatic patients with internal carotid stenosis, who underwent an endarterectomy procedure. Each patient had their carotid artery sampled in order to find C. pneumoniae DNA using the nested PCR method and some cytokines (TGF-ß, VEGF, FGF, TNF-α) using immunohistochemical examination. The control group consisted of 20 young organ donors who had been diagnosed with brain death and who had their healthy carotid artery harvested. Analogous genetic and immunohistochemical tests were performed. RESULTS: We did not confirm the presence of either cytokines or C. pneumoniae in the healthy carotid arteries. The presence of FGF was probably due to intima fibroblast activity, which is responsible for elastin and collagen synthesis for the extracellular matrix. C. pneumoniae was discovered in 68% of patients with carotid plaques. Three cytokines (TGF-ß, FGF, TNF-α) were detected in atherosclerotic internal carotid arteries as well. CONCLUSION: Chronic infection by C. pneumoniae may exacerbate carotid plaque development and may lead to its destabilization.

Bacteria present in carotid arterial plaques are found as biofilm deposits which may contribute to enhanced risk of plaque rupture.

UNLABELLED: Atherosclerosis, a disease condition resulting from the buildup of fatty plaque deposits within arterial walls, is the major underlying cause of ischemia (restriction of the blood), leading to obstruction of peripheral arteries, congestive heart failure, heart attack, and stroke in humans. Emerging research indicates that factors including inflammation and infection may play a key role in the progression of atherosclerosis. In the current work, atherosclerotic carotid artery explants from 15 patients were all shown to test positive for the presence of eubacterial 16S rRNA genes. Density gradient gel electrophoresis of 5 of these samples revealed that each contained 10 or more distinct 16S rRNA gene sequences. Direct microscopic observation of transverse sections from 5 diseased carotid arteries analyzed with a eubacterium-specific peptide nucleic acid probe revealed these to have formed biofilm deposits, with from 1 to 6 deposits per thin section of plaque analyzed. A majority, 93%, of deposits was located proximal to the internal elastic lamina and associated with fibrous tissue. In 6 of the 15 plaques analyzed, 16S rRNA genes from Pseudomonas spp. were detected. Pseudomonas aeruginosa biofilms have been shown in our lab to undergo a dispersion response when challenged with free iron in vitro. Iron is known to be released into the blood by transferrin following interaction with catecholamine hormones, such as norepinephrine. Experiments performed in vitro showed that addition of physiologically relevant levels of norepinephrine induced dispersion of P. aeruginosa biofilms when grown under low iron conditions in the presence but not in the absence of physiological levels of transferrin. IMPORTANCE: The association of bacteria with atherosclerosis has been only superficially studied, with little attention focused on the potential of bacteria to form biofilms within arterial plaques. In the current work, we show that bacteria form biofilm deposits within carotid arterial plaques, and we demonstrate that one species we have identified in plaques can be stimulated in vitro to undergo a biofilm dispersion response when challenged with physiologically relevant levels of norepinephrine in the presence of transferrin. Biofilm dispersion is characterized by the release of bacterial enzymes into the surroundings of biofilm microcolonies, allowing bacteria to escape the biofilm matrix. We believe these enzymes may have the potential to damage surrounding tissues and facilitate plaque rupture if norepinephrine is able to stimulate biofilm dispersion in vivo. This research, therefore, suggests a potential mechanistic link between hormonal state and the potential for heart attack and stroke.

Early atherosclerotic plaques show evidence of infection by Chlamydia pneumoniae.

Chlamydia pneumoniae (Cpn) could play an important role in the development of atherosclerosis. Cpn interferes with HIF-1alpha regulation in infected host cells during intracellular replication in hypoxia. We obtained carotid artery specimens with low (n=38), high (n=25) levels of stenosis and 10 middle cerebral arteries. Fifty eight percent of the carotids with low levels of stenosis showed evidence of the viable organism. Ninety one percent of the positive results were derived from pre-atheromatous lesions. Only 12 percent of plaques removed at endarterectomy showed the presence of Cpn DNA. All middle cerebral arteries failed to show evidence of live Chlamydia. Ninety one percent of sera from 22 endarterectomy patients failed to show the presence of Cpn antibodies. Immunohistology of carotid arteries with low levels of stenosis was used to confirm the presence of HIF-1alpha in infected specimens and showed a correlation between the over-expression of HIF-1alpha and Cpn in the plaque (p less than 0.05). Cpn might play an important role in activation and development of the initial stages of atherosclerotic lesions.

[Pecularities of atherosclerosis developed after chlamidial infection and behaviour of some hematological and biochemical parameters].

The aim of our studies was to create diagnostically informative criteria of hematologic and biochemical analyses and their combinations as well as to establish relation between Chlamidia Pneumoniae A and atherosclerosis. This is to be done for the purpose of establishing the entire pathogenesis carotid and coronary atherosclerosis and inflammation. The investigation of Chlamidia Pneumoniae infection in proved to be a possible risk factor for the carotid atherosclerosis developed in adults that makes the course of atherosclerosis heavier. As a result of the atherosclerotic process, changes in the blood laboratory parameters were expressed more cases of pneumonia in the children.

Combined endovascular and open operative approach for mycotic carotid aneurysm.

Mycotic aneurysms of the extracranial carotid artery are rare and warrant surgical intervention. Management involves open and endovascular approaches. We report the case of a 67-year-old woman with an Escherichia coli soft-tissue infection of the right retropharyngeal space and subsequent mycotic carotid aneurysm and thrombosis of the internal jugular vein. The patient presented with a pulsatile mass and right middle cerebral artery stroke. Our surgical management involved coil embolization of the aneurysm to provide for vascular control, with resection of the common carotid artery, internal carotid artery, and extracranial carotid artery branches, along with the internal jugular vein.

Infectious burden and carotid plaque thickness: the northern Manhattan study.

BACKGROUND AND PURPOSE: The overall burden of prior infections may contribute to atherosclerosis and stroke risk. We hypothesized that serological evidence of common infections would be associated with carotid plaque thickness in a multiethnic cohort. METHODS: Antibody titers to 5 common infectious microorganisms (ie, Chlamydia pneumoniae, Helicobacter pylori, cytomegalovirus, and herpesvirus 1 and 2) were measured among stroke-free community participants and a weighted index of infectious burden was calculated based on Cox models previously derived for the association of each infection with stroke risk. High-resolution carotid duplex Doppler studies were used to assess maximum carotid plaque thickness. Weighted least squares regression was used to measure the association between infectious burden and maximum carotid plaque thickness after adjusting for other risk factors. RESULTS: Serological results for all 5 infectious organisms were available in 861 participants with maximum carotid plaque thickness measurements available (mean age, 67.2+/-9.6 years). Each individual infection was associated with stroke risk after adjusting for other risk factors. The infectious burden index (n=861) had a mean of 1.00+/-0.35 SD and a median of 1.08. Plaque was present in 52% of participants (mean, 0.90+/-1.04 mm). Infectious burden was associated with maximum carotid plaque thickness (adjusted increase in maximum carotid plaque thickness 0.09 mm; 95% CI, 0.03 to 0.15 mm per SD increase of infectious burden). CONCLUSIONS: A quantitative weighted index of infectious burden, derived from the magnitude of association of individual infections with stroke, was associated with carotid plaque thickness in this multiethnic cohort. These results lend support to the notion that past or chronic exposure to common infections, perhaps by exacerbating inflammation, contributes to atherosclerosis. Future studies are needed to confirm this hypothesis and to define optimal measures of infectious burden as a vascular risk factor.

Prevalence of Chlamydophila pneumoniae is higher in aorta and coronary artery than in carotid artery of coronary artery disease patients.

Coronary artery disease (CAD) is a public health problem accounting for an estimated one-third of deaths overall. A potential link between infectious agents and atherosclerosis has been suggested. Data obtained from several seroepidemiological studies have suggested that infection with Chlamydophila pneumoniae, Helicobacter pylori, cytomegalovirus and herpes simplex virus-1 can initiate or maintain the atherosclerotic process. However, there is no single study in which multiple infectious agents have been detected together in different vascular locations in the same population. This would help in determining if there is any leading pathogen in atheromatous plaques of CAD patients. Hence, we screened for C. pneumoniae, H. pylori, CMV and HSV-1 in different vascular locations of CAD patients using quantitative real-time (RT) PCR. We performed multiplex RT-PCR for detecting pathogens, viz. C. pneumoniae, H. pylori, CMV and HSV-1 in different vascular locations of CAD patients. Percent positivity scores for C. pneumoniae, H. pylori, CMV and HSV-1 in different vascular locations were as follows: aorta (64.7, 35.3, 11.7 and 11.7 respectively); carotid (27.2, 27.2, 9 and 0 respectively); coronary artery (58.3, 33.3, 16.6 and 8.3 respectively). Combined positivity for C. pneumoniae (C. pneumoniae IgA and RT-PCR for C. pneumoniae) was the highest compared with all other groups. Aorta and coronary artery were more susceptible to these pathogens as compared with carotid artery. Moreover, CAD patients' characteristics were associated with C. pneumoniae positivity (C. pneumoniae IgA and RT-PCR), suggesting thereby that C. pneumoniae may have caused chronic persistent infection in CAD.

Real-time polymerase chain reaction and laser capture microdissection: an efficient combination tool for Chlamydophila pneumoniae DNA quantification and localization of infection in atherosclerotic lesions.

Chlamydophila pneumoniae has been implicated in atherosclerosis, but the role of this obligate intracellular pathogen in the development of the above pathology is still unclear. In particular, its presence and quantitative distribution within lesional areas has not yet been defined. We studied 18 carotid biopsies obtained from patients undergoing endoartherectomy. By laser microdissection (LCM), two different sites (intra-plaque and plaque-adjacent areas) were taken from each lesion, and the presence and quantity of the pathogen DNA were determined by real-time polymerase chain reaction (Real-time PCR). A total of 8 plaques, exclusively from patients with unstable angina, were positive in real-time PCR. The bacterial DNA was detected in both lesional areas of 3 plaques which contained the highest number of DNA copies (1,900 to 2,200 copy numbers), while C. pneumoniae DNA was detected only in the intra-plaque area of the other 5 positive (500 to 1,600 copy numbers). No C. pneumoniae DNA was found in the other 10 plaques of which 6 were from patients with unstable angina and 4 from stable angina patients. No DNA from Helicobacter pylori or Cytomegalovirus was found in any plaque. This is the first report where both the target lesion and an adjacent reference site were evaluated for the presence of C. pneumoniae DNA by the combination of LCM and Real-time PCR assays. The integration of these two methodologies offer an excellent tool for in situ studies and may help to elucidate the putative role of C. pneumoniae in atherosclerosis.